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Abstract
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Protocol
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Biology

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Published: September 28, 2021
doi:
10.3791/62882
, , , , , ,
1Department of Entomology,National Chung Hsing University, 2Department of Agricultural biology,Jeonbuk National University, 3Department of Agricultural Convergence Technology,Jeonbuk National University

Summary

在这里，我们提出了一种基于黄粉虫（Tenebrio molitor）诱饵系统的方案，该系统用于从土壤样品中分离和选择昆虫致病真菌（EPF）。采用有效的分生孢子数（ECN）公式，根据生理特性选择高耐压EPF，用于野外害虫微生物防治。

Abstract

昆虫致病真菌（EPF）是用于综合虫害管理的微生物控制剂之一。为了控制本地或入侵性害虫，隔离和选择本地EPF非常重要。因此，本研究采用土壤饵料法与昆虫饵料（黄粉虫、 Tenebrio molitor）系统相结合，并作了一些修改。然后将分离出的EPF对农业害虫 Spodoptera litura进行毒力测试。此外，对潜在的EPF菌株进行了形态学和分子鉴定。此外，对有前景的EPF菌株进行了分生孢子生产和耐热性测定并进行了比较;这些数据被进一步替换为有效分生孢子数（ECN）公式，用于实验室排名。土壤饵料-黄粉虫系统和ECN配方可以通过替代昆虫种类和整合更多胁迫因素来改善商业化和田间应用的评价。该协议为EPF选择提供了一种快速有效的方法，并将改进生物控制剂的研究。

Introduction

目前，昆虫病原真菌（EPF）广泛用于农业，森林和园艺害虫的微生物控制。EPF的优点是宿主范围广，环境适应性好，环保性好，可与其他化学品配合使用，显示出病虫害综合治理的协同效应1，2。对于作为害虫控制剂的应用，有必要从患病昆虫或自然环境中分离出大量EPF。

从宿主中对这些生物体进行采样有助于了解EPF在自然宿主中的地理分布和流行率3，4，5。然而，真菌感染昆虫的收集通常受到环境因素和野外昆虫种群的限制4。考虑到昆虫宿主会在EPF感染后死亡，然后落入土壤中，从土壤样本中分离EPF可能是一种稳定的资源3，6。例如，已知腐生植物使用死亡宿主作为其生长资源。土壤饵料和选择性介质系统已被广泛用于土壤中EPF的检测和分离3，4，7，8，9，10。

在选择性培养基方法中，将稀释的土壤溶液接种到含有广谱抗生素（例如氯霉素，四环素或链霉素）的培养基上以抑制细菌的生长2，3，9，11。然而，据报道，这种方法可能会扭曲菌株的多样性和密度，并可能导致对许多微生物群落的高估或低估6。此外，分离的菌株致病性较低，并且在分离过程中与腐生植物竞争。很难从稀释的土壤溶液中分离出EPF3。土壤诱饵法不使用选择性培养基，而是将EPF与受感染的死昆虫分离出来，这些害虫可以储存2-3周，从而提供更有效和标准的EPF分离方法3，4，7，6。由于该方法易于操作，因此可以低成本分离出多种致病菌株4。因此，它被许多研究人员广泛使用。

在比较了不同类型的昆虫诱饵系统后， 贝氏 蜈蚣和 苜蓿 是最常见的EPF物种，存在于属于半翅目，鳞翅目，布拉泰拉和鞘翅目昆虫中6，12，13，14。在这些昆虫诱饵中，与其他昆虫相比， 鳞 翅目（鳞翅目）和 鞘 翅目（鞘翅目）显示出更高的 Beauveria 和 Metarhizium spp.的回收率。因此， G. mellonella 和 T. molitor 通常用于昆虫诱饵。多年来，美国农业部（USDA）建立了一个EPF图书馆（农业研究服务EPF菌种馆藏，ARSEF），其中包含各种各样的物种，包括4081种 Beauveria spp.，18种 Clonostaches spp.，878种 冬虫夏草 属，2473种 Metarhizium spp.，226种 Purpureocillium spp.， 和13种 Pochonia spp.等15。另一个EPF图书馆由美国佛蒙特大学的昆虫学研究实验室（ERL）建造，历时30年。它包括来自美国、欧洲、亚洲、非洲和中东的 1345 株 EPF16。

为了控制台湾的本地或入侵害虫，需要隔离和选择本地EPF。因此，在该协议中，我们修改并描述了土壤诱饵方法的程序，并将其与昆虫诱饵（黄粉虫， Tenebrio molitor）系统相结合17。基于该协议，建立了一个EPF库。对初步EPF分离株进行了两轮筛选（接种量定量）。EPF分离株显示出对昆虫的致病性。对潜在菌株进行形态学和分子鉴定，并通过耐热性和分生孢子生产测定法进一步分析。此外，还提出了有效分生孢子数（ECN）的概念。采用ECN公式和主成分分析（PCA），在模拟环境压力下对潜在菌株进行分析，完成EPF库的建立和筛选过程。随后，对目标害虫（例如， Spodoptera litura）测试了有希望的EPF菌株的致病性。目前的协议将耐热性和分生孢子生产数据整合到ECN公式和PCA分析中，可用作EPF相关研究的标准排名系统。

Protocol

注：整个流程图如图 1所示。 1. 潜在昆虫致病真菌（EPF）的分离和选择 收集土壤样本 去除1厘米的表层土壤，然后使用铲子从每个采样点收集5-10厘米深度内的土壤。注：采样点为山区、森林或人口稀少的地区，以避免人工喷洒的EPF菌株受到污染。确保土壤样品采集区域表面覆盖杂草。干燥的土壤或潮湿的土壤不适合这个实验。 ?…

Representative Results

潜在昆虫病原真菌（EPF）的分离和选择通过使用Tenebrio molitor介导的昆虫致病真菌（EPF）文库构建方法，可以排除没有杀虫活性的真菌数量;因此，可以大大提高EPF的隔离效率和选择。在该方法的应用过程中，记录了采样地点，土壤样品和真菌发芽率的信息（表2）。根据我们之前的数据，从172个土壤样品中共获得101个真菌分离物，表明分?…

Discussion

昆虫致病真菌（EPF）已被用于昆虫控制。有几种方法可以分离，选择和鉴定EPF30，31，32。比较不同种类的昆虫诱饵方法，在昆虫诱饵中常见于贝氏苜蓿和茴香6、12、13、14。在这些昆虫诱饵中，Galleria mellonella和<…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了科学和技术部（MOST）的资助109-2313-B-005 -048 -MY3的支持。

Materials

Agar Bacteriological grade BIOMAN SCIENTIFIC Co., Ltd. AGR001 Suitable in most cell culture/molecular, biology applications.
AGAROSE, Biotechnology Grade BIOMAN SCIENTIFIC Co., Ltd. AGA001 For DNA electrophoresis.
BioGreen Safe DNA Gel Buffer BIOMAN SDB001T
Brass cork borer Dogger D89A-44001
Canon kiss x2 Canon EOS 450D For record strain colony morphology
Constant temperature incubator Yihder Co., Ltd. LE-509RD Fungal keeping.
cubee Mini-Centrifuge GeneReach MC-CUBEE
DigiGel 10 Digital Gel Image System TOPBIO DGIS-12S
Finnpipette F2 0.2 to 2 µL Pipette Thermo Scientific 4642010
Finnpipette F2 1 to 10 µL Pipette Thermo Scientific 4642030
Finnpipette F2 10 to 100 µL Pipette Thermo Scientific 4642070
Finnpipette F2 100 to 1000 µL Pipette Thermo Scientific 4642090
Finnpipette F2 2 to 20 µL Pipette Thermo Scientific 4642060
Finnpipette F2 20 to 200 µL Pipette Thermo Scientific 4642080
GeneAmp PCR System 9700 Applied Biosystems 4342718
GenepHlow Gel/PCR Kit Geneaid DFH100
Genius Dry Bath Incubator Major Science MD-01N
Graduated Cylinder Custom A 100mL SIBATA SABP-1195906 Measure the volume of reagents.
Hand tally counter SDI NO.1055
Hemocytometer bioman AP-0650010 Calculate the number of spore
Inoculating loop Dogger D8GA-23000
lid IDEAHOUSE RS92004
Micro cover glass MUTO PURE CHEMICALS CO.,LTD 24241
Microscope imaging system SAGE VISION CO.,LTD SGHD-3.6C
Microscope Slides DOGGER DG75001-07105
Mupid-2plus DNA Gel Electrophoresis ADVANCE AD110
Nikon optical microscope SAGE VISION CO.,LTD Eclipse CI-L
Plastic cup IDEAHOUSE CS60016
Presto Mini gDNA Yeast Kit Geneaid GYBY300 Fungal genomic DNA extraction kit
Sabouraud Dextrose Broth (Sabouraud Liquid Medium) HiMedia Leading BioSciences Company M033 Used for cultivation of yeasts, moulds and aciduric microorganisms.
Scalpel Blade No.23 Swann-Morton 310
Scalpel Handle No.4 AGARWAL SURGICALS SSS -FOR-01-91
Shovel Save & Safe A -1580242 -00
Silwet L-77 bioman(phytotech) S7777 Surfactant
Sorvall Legend Micro 17 Microcentrifuge Thermo Scientific 75002403
Steel Tweezers SIPEL ELECTRONIC SA GG-SA
Sterile Petri Dish BIOMAN SCIENTIFIC Co., Ltd. 1621 Shallow cylindrical containers with fitted lids, specifically for microbiology or cell culture use.
ThermoCell MixingBlock BIOER MB-101
Tween 80 FUJIFILM Wako Pure Chemical Corporation 164-21775
TwinGuard ULT Freezer Panasonic Healthcare Holdings Co., Ltd. MDF-DU302VX -80°C sample stored.
Vertical floor type cabinet Chih Chin BSC-3 Fungal operating culturing.
Vortex Genie II Scientific SIG560
Zipper storage bags Save & Safe A -1248915 -00
100 bp DNA Ladder Geneaid DL007
-20°C Freezer FRIGIDAIRE Frigidaire FFFU21M1QW -20°C sample and experimental reagents stored.
2X SuperRed PCR Master Mix TOOLS TE-SR01
50X TAE Buffer BIOMAN TAE501000
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Tags

Entomopathogenic FungiSoil SamplesInsect PestsIsolationVirulencePathogenicity ScreeningTenebrio MolitorSabouraud Dextrose AgarThermotoleranceConidia Production
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Liu, Y., Ni, N., Chang, J., Li, Y., Lee, M. R., Kim, J. S., Nai, Y. Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests. J. Vis. Exp. (175), e62882, doi:10.3791/62882 (2021).
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